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Applied and Environmental Microbiology, December 2007, p. 7542-7547, Vol. 73, No. 23
0099-2240/07/$08.00+0     doi:10.1128/AEM.01023-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Identification of the Propionicin F Bacteriocin Immunity Gene (pcfI) and Development of a Food-Grade Cloning System for Propionibacterium freudenreichii ^,

Laboratory of Microbial Gene Technology and Food Microbiology, Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences (UMB), P.O. Box 5003, N-1432 Ås, Norway

Received 8 May 2007/ Accepted 30 September 2007

This report describes the first functional analysis of a bacteriocin immunity gene from Propionibacterium freudenreichii and its use as a selection marker for food-grade cloning. Cloning of the pcfI gene (previously orf5 [located as part of the pcfABC propionicin F operon]) rendered the sensitive host 1,000-fold more tolerant to the propionicin F bacteriocin. The physiochemical properties of the 127-residue large PcfI protein resemble those of membrane-bound immunity proteins from bacteriocin systems found in lactic acid bacteria. The high level of immunity conferred by pcfI allowed its use as a selection marker for plasmid transformation in P. freudenreichii. Electroporation of P. freudenreichii IFO12426 by use of the pcfI expression plasmid pSL102 and propionicin F selection (200 bacteriocin units/ml) yielded 10⁷ transformants/µg DNA. The 2.7-kb P. freudenreichii food-grade cloning vector pSL104 consists of the pLME108 replicon, a multiple cloning site, and pcfI expressed from the constitutive P_pampS promoter for selection. The pSL104 vector efficiently facilitated cloning of the propionicin T1 bacteriocin in P. freudenreichii. High-level propionicin T1 production (640 BU/ml) was obtained with the IFO12426 strain, and the food-grade propionicin T1 expression plasmid pSL106 was maintained by 91% of the cells over 25 generations in the absence of selection. To the best of our knowledge this is the first report of an efficient cloning system that facilitates the generation of food-grade recombinant P. freudenreichii strains.

Published ahead of print on 12 October 2007.

Supplemental material for this article may be found at http://aem.asm.org/.

This article has been cited by other articles:

• Faye, T., Asebo, A., Salehian, Z., Langsrud, T., Nes, I. F., Brede, D. A. (2008). Construction of a Reporter Vector System for In Vivo Analysis of Promoter Activity in Propionibacterium freudenreichii. Appl. Environ. Microbiol. 74: 3615-3617 [Abstract] [Full Text]